Experiment
1,25-Dihydroxyvitamin D (1,25[OH]2D) regulates calcium (Ca), phosphate, and bone metabolism. Serum 1,25(OH)2D levels are reduced by low vitamin D status and high fibroblast growth factor 23 (FGF23) levels and increased by low Ca intake and high PTH levels. In the current study two genetically diverse populations of inbred mice are used to characterize the impact of natural genetic variation on basal serum 1,25(OH)2D levels on the response of serum 1,25(OH)2D levels to dietary Ca restriction and on the basal serum levels of 25(OH)D and FGF23.
Study 1 examined a genetically diverse population of 11 inbred mouse lines: 129S1/SV1mJ, A/J, AKR/J, C3H/HeJ, C57BL/6J (B6), CAST/EiJ, CBA/J, DBA/2J (DBA), PWK/PhJ, SWR/J, and WSB/EiJ. Intestinal Ca absorption and bone-related phenotypes for this population have been reported previously (17, Repogle et al, 2014).
Study 2 was a forward genetic linkage mapping study to identify quantitative trait loci (QTLs) influencing serum vitamin D metabolites using 51 lines from the BXD (C57BL/6J x DBA/2J) recombinant inbred (RI) panel. Genetic mapping revealed many loci controlling 1,25(OH)2D and the RCR as well as 25(OH)D and FGF23.
Male mice from 51 lines of the BXD recombinant inbred (RI) panel were fed diets with either 0.5% (adequate) or 0.25% (low) calcium from 4 to 12 weeks of age (n=8 per line per diet). The low-Ca diet reflects the low Ca intake commonly seen in the United States (ie, 50% of the requirement), and it induces an adaptive increase in Ca absorption (90%) in C57BL/6J mice. At 12 weeks of age the mice were fasted overnight and then anesthetized, then euthanized, and the left kidney removed. Serum was prepared and both kidney and serum were frozen. Commercial RIAs were used to measure the serum levels of 25(OH)D and 1,25(OH)2D. Intact FGF23 was measured in the serum of mice fed the 0.5% Ca diet using a commercial ELISA.
Statistical analyses were conducted using SAS Enterprise Guide 4.2 (SAS Institute Inc). Analysis of covariance was used to test for the main effects of genetic background (ie, line) and diet as well as a line-by-diet interaction while controlling for the effect of body weight (BW), femur length, or serum analysis kit as covariates. The confounding effect of covariates was removed by linear regression, and residuals were used for linkage mapping. Marker information and BXD genotypes were downloaded from The GeneNetwork (http://www.genenetwork.org/genotypes/BXD.geno), and the genetic location of each marker was updated using the Mouse Map Converter tool at the Jackson Labs Center for Genome Dynamics (http://cgd.jax.org/mousemapconverter/).
Composite interval mapping (CIM) was conducted using Windows QTL Cartographer version 2.5_011. . CIM was carried out using a Haldane map function, 2 cM walking speed, and a 10-cM window. Each diet group and the response to dietary Ca restriction (RCR, 1,25[OH]2D
only) were mapped separately. Five hundred permutations were used to determine the significance for each analysis.
Table 1: (QTL locations were based on GRCm38/mm10.)
QTLs for serum 1,25(OH)2D were significant in at least one or both diets or for the RCR.
QTL V125Dq1 (vitamin D active form serum level QTL) mapped to Chromosome 1 in the 0.5% calcium diet with a peak at 21.6 cM (43.0 Mb), LOD=3.5, accounting for 6.2 % of trait variance.
QTL V125Dq2 (vitamin D active form serum level QTL 2) mapped to Chromosome 1 in the 0.25% calcium diet with a peak at 15.2 cM (36.3 Mb), LOD=2.93, accounting for 3.3 % of trait variance. The C57BL/6J allele conferred influence at both Chr 1 QTL.
QTL V125Dq3 (vitamin D active form serum level QTL 3) mapped to Chromosome 1 in the RCR analysis (the response of serum 1,25(OH)2D levels in dietary calcium restriction) with a peak at 61.6 cM (140.0 Mb), LOD=2.93, accounting for 12.2 percent of trait variance. The C57BL/6J allele conferred influence at all three QTL.
QTL V125Dq4 (vitamin D active form serum level QTL 4) mapped to Chromosome 3 in the 0.5% calcium diet with a peak at 33.7 cM (76.0 Mb), LOD=5.0, accounting for 9.7 percent of trait variance. The C57BL/6J allele was the influential allele at this locus.
QTL V125Dq5 (vitamin D active form serum level QTL 5) mapped to Chromosome 7 in the 0.5% calcium diet with a peak at 34.4 cM (62.6 Mb), LOD=2.5, accounting for 3.5% of trait variance.
QTL V125Dq6 (vitamin D active form serum level QTL 6) mapped to Chromosome 7 in the 0.25% calcium diet with a peak at 38.1 cM (69.5 Mb), LOD=3.7, accounting for 5.9% of trait variance. The C57BL/6J allele was the influential allele at both Chr 7 loci.
QTL V125Dq7 (vitamin D active form serum level QTL 7) mapped to Chromosome 9 in the 0.5 % calcium diet with a peak at 18.1 cM (33.1 Mb), LOD=12.7, accounting for 34.1 percent of trait variance.
QTL V125Dq8 (vitamin D active form serum level QTL 8) mapped to Chromosome 9 in the 0.25% calcium diet with a peak at 18.1 cM (33.1 Mb), LOD=10.5, accounting for 33.7 percent to trait variation. The C57BL/6J allele was the influential allele at both Chr 9 loci.
QTL V125Dq9 (vitamin D active form serum level QTL 9) mapped to Chromosome 10 in the RCR analysis with a peak at 61.7 cM (114.7 Mb), LOD=4.2, accounting for 19% of trait variance. The DBA/2J allele was the influential allele at this locus.
QTL V125Dq10 (vitamin D active form serum level QTL 10) mapped to Chromosome 13 in the 0.05% calcium diet with a peak at 49.3 cM (94.9 Mb), LOD=5.3, accounting for 10.4% of trait variation. The C57BL/6J allele was the influential allele at this locus.
QTL V125Dq11 (vitamin D active form serum level QTL 11) mapped to Chromosome 15 in the 0.25% calcium diet with a peak at 51.3 cM (96.0 Mb), LOD=7.9, accounting for 15% of trait variance. The DBA/2J allele was the influential allele at this locus.
QTL V125Dq12 (vitamin D active form serum level QTL 12) mapped to Chromosome 18 in the RCR analysis with a peak at 25.0 cM (47.1 Mb), LOD=3.6, accounting for 21.0% of trait variance. The DBA/2J allele was the influential allele at this locus.
QTL V125Dq13 (vitamin D active form serum level QTL 13) mapped to Chromosome 18 in the 0.5% calcium diet with a peak at 47.9 cM (73.3 Mb), LOD=5.6, accounting for 11.0% of trait variance.
QTL V125Dq14 (vitamin D active form serum level QTL 14) mapped to Chromosome 18 in the 0.25% calcium diet with a peak at 48.77 cM (73.4 Mb), LOD=6.4, accounting for 11.7% of trait variance. The C57BL/6J allele was the influential allele at both b loci.
QTL influencing 25(OH)D serum levels:
QTL V25Dq1 (vitamin D inactive form serum level QTL 1) mapped to Chromosome 1 in the 0.5% calcium diet with a peak at 95.4 (189.9 Mb), LOD=4.1, accounting for 17.2% of trait variance.
QTL V25Dq2 (vitamin D inactive form serum level QTL 2) mapped to Chromosome 1 in the 0.25% calcium diet with a peak at 95.4 (189.9 Mb), LOD=4.9, accounting for 16.7% of trait variance. The C57BL/6J allele was influential at both loci.
QTL V25Dq3 (vitamin D inactive form serum level QTL 3) mapped to Chromosome 2 in the 0.5% calcium diet with a peak at 71.8 cM (146.5 Mb), LOD=2.89, accounting for 11.4% of trait variance. The DBA/2J allele was the influential allele at this locus.
QTL V25Dq4 (vitamin D inactive form serum level QTL 4) mapped to Chromosome 6 in the 0.5% calcium diet with a peak 28.2 cM (58.6 Mb), LOD=4.5, accounting for 19.2% of trait variance.
QTL V25Dq5 (vitamin D inactive form serum level QTL 5) mapped to Chromosome 6 in the 0.25% calcium diet with a peak at 24.0 cM (49.7 Mb), LOD=2.1, accounting for 5.2% of trait variance. The C57BL/6J allele was the influential allele at both loci.
QTL V25Dq6 (vitamin D inactive form serum level QTL 6) mapped to Chromosome 9 in the 0.25% calcium diet with a peak at 56.62 cM (104.8 Mb), LOD=2.6, accounting for 7.9% of trait variance. The C57BL/6J allele was the influential allele at this locus.
QTL V25Dq7 (vitamin D inactive form serum level QTL 7) mapped to Chromosome 13 in the 0.25% calcium diet with a peak at 14.4 cM (36.4 Mb), LOD=6.3, accounting for 23.4 of trait variance. The C57BL/6J allele was the influential allele at this locus.
QTL V25Dq8 (vitamin D inactive form serum level QTL 8) mapped to Chromosome 15 in the 0.25% calcium diet with a peak at 37.57 cM (78.7 Mb), LOD=4.1, accounting for 13.7% of trait variance. The C57BL/6J allele was the influential allele at this locus.
QTL V25Dq9 (vitamin D inactive form serum level QTL 9) mapped to Chromosome X in the 0.25% calcium diet with a peak at 0.01 cM (0.0 Mb), LOD=2.5, accounting for 7.8% of trait variance. The C57BL/6J allele was the influential allele at this locus.
QTL influencing FGF23 serum levels:
QTL Fgf23lq1 (FGF23 serum level QTL 1) mapped to Chromosome 4 in the 0.50% calcium diet with a peak at 54.5 cM (118.3 Mb), LOD=2.9, accounting for 11.7% of trait variation.
QTL Fgf23lq2 (FGF23 serum level QTL 2) mapped to Chromosome 8 in the 0.50% calcium diet with a peak at 60.3 cM (113.9 Mb), LOD=2.8, accounting for 11.2% of trait variation. The C57BL/6J allele was the influential allele at both loci.
QTL Fgf23lq3 (FGF23 serum level QTL 3) mapped to Chromosome 14 in the 0.50% calcium diet with a peak at 34.8 cM (70.7 Mb), LOD=3.6, accounting for 15.0% of trait variation.
QTL Fgf23lq4 (FGF23 serum level QTL 4) mapped to Chromosome 18 in the 0.50% calcium diet with a peak at 47.9 cM (73.3 Mb), LOD=4.1, accounting for 16.4% of trait variation. The DBA/2J allele was the influential allele at both the Chr 14 and the Chr 18 loci.
Table 2 lists a summary of potential candidate genes based on nonsynonymous SNPs; deleterious nonsynonymous SNP changes; overlapping eQTL; mRNA levels significantly correlated with the respective phenotype.
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